Human mesenchymal stromal cells inhibit platelet activation and aggregation involving CD73-converted adenosine.

BACKGROUND: Mesenchymal stromal cells (MSCs) are promising cell therapy candidates. Clinical application is considered safe. However, minor side effects have included thromboembolism and instant blood-mediated inflammatory reactions suggesting an effect of MSC infusion on hemostasis. Previous studies focusing on plasmatic coagulation as a secondary hemostasis step detected both procoagulatory and anticoagulatory activities of MSCs. We now focus on primary hemostasis and analyzed whether MSCs can promote or inhibit platelet activation. METHODS: Effects of MSCs and MSC supernatant on platelet activation and function were studied using flow cytometry and further platelet function analyses. MSCs from bone marrow (BM), lipoaspirate (LA) and cord blood (CB) were compared to human umbilical vein endothelial cells or HeLa tumor cells as inhibitory or activating cells, respectively. RESULTS: BM-MSCs and LA-MSCs inhibited activation and aggregation of stimulated platelets independent of the agonist used. This inhibitory effect was confirmed in diagnostic point-of-care platelet function analyses in platelet-rich plasma and whole blood. Using inhibitors of the CD39-CD73-adenosine axis, we showed that adenosine produced by CD73 ectonucleotidase activity was largely responsible for the LA-MSC and BM-MSC platelet inhibitory action. With CB-MSCs, batch-dependent responses were obvious, with some batches exerting inhibition and others lacking this effect. CONCLUSIONS: Studies focusing on plasmatic coagulation suggested both procoagulatory and anticoagulatory activities of MSCs. We now show that MSCs can, dependent on their tissue origin, inhibit platelet activation involving adenosine converted from adenosine monophosphate by CD73 ectonucleotidase activity. These data may have strong implications for safety and risk/benefit assessment regarding MSCs from different tissue sources and may help to explain the tissue protective mode of action of MSCs. The adenosinergic pathway emerges as a key mechanism by which MSCs exert hemostatic and immunomodulatory functions.

Extension of platelet shelf life from 4 to 5 days by implementation of a new screening strategy in Germany.

BACKGROUND: The Paul-Ehrlich-Institute analysed all fatalities due to bacterial infections between 1997 and 2007. Thereafter, the platelet shelf life was reduced to a maximum of 4 days after blood donation because the majority of all cases of severe transfusion-transmitted bacterial infections occurred with day 5 platelets. The current study compares the analytical sensitivity and the diagnostic specificity of four rapid bacterial detection procedures. METHODS: Nine transfusion-relevant bacterial strains were spiked in pooled platelets or apheresis platelets at a low concentration (10 CFU/bag). Samples were collected after day 3, day 4 and day 5 and investigated by four rapid bacterial detection methods (modified BacT/ALERT, Bactiflow, FACS method and 16s DNA PCR methods). RESULTS: Seven out of nine bacterial strains were adequately detected by BacT/ALERT, Bactiflow and PCR in apheresis platelets and pooled platelets after sample collection at day 3, day 4 and day 5. For three bacterial strains, analytical sensitivity was reduced for the FACS method. Two bacterial strains did not grow under the storage conditions in either pooled or apheresis platelets. CONCLUSIONS: A late sample collection on day 3, day 4 or day 5 after blood donation in combination with a rapid bacterial detection method offers a new opportunity to improve blood safety and reduce errors due to sampling., BacT/ALERT, Bactiflow or 16s ID-NAT are feasible for late bacterial screening in platelets may provide data which support the extension of platelet shelf life in Germany to 5 days.

Pathogen reduction technology (Mirasol) treated single-donor platelets resuspended in a mixture of autologous plasma and PAS.

BACKGROUND AND OBJECTIVES: Mirasol pathogen reduction technology (PRT) for platelet concentrates uses riboflavin and ultraviolet light. Previously, we described increased metabolism and activation for PRT platelets stored in 100% plasma. To improve platelet quality, we resuspended platelets in a mixture of plasma and platelet additive solution (PAS). MATERIALS AND METHODS: Single-donor platelets were resuspended in plasma and split into an untreated control and a PRT-treated single product. One hundred and fifty millilitre PAS (SSP+) was added to both. Over 7 days, we assayed pH, glucose consumption-, lactate production rate and CD62p with and without TRAP. RESULTS: On day 5, PRT units showed a significantly lower pH (7.087 +/- 0.105 vs. 7.288 +/- 0.200) accompanied by a higher lactate production (0.104 +/- 0014 vs. 0.063 +/- 0.017 mmol/10(12)/h) and glucose consumption rate (0.039 +/- 0005 vs. 0.028 +/- 0.009 mmol/10(12) platelets/h). CD62p expression was higher in treated units (44.5 +/- 13.0 vs. 16.5 +/- 7.6%). CONCLUSION: In comparison to PRT platelets resuspended in 100% plasma, a mixture of plasma and PAS improves pH and platelet metabolism but not platelet activation. Prolonged shelf-life for up to 7 days may be possible

Sensitivity and specificity of Anti-HBc screening assays--which assay is best for blood donor screening?

Compared to HIV and hepatitis C virus, the residual infectious risk of hepatitis B virus (HBV) posed by blood products is about 10 times higher. In addition to HBsAg testing, screening for anti-HBc was recommended by the German Advisory Committee Blood in March 2005. Prevalence of anti-HBc in German blood donors was investigated at five test sites located in different geographic regions. In total, 12,000 blood donors were screened for anti-HBc by PRISM HBcore, and a statistically representative number of these were tested with Abbott Murex anti-HBc total, bioMérieux Hepanostika anti-HBc uniform, Bio-Rad Monolisa anti-HBc PLUS and Dade Behring Enzygnost anti-HBc. Anti-HBc repeat reactive samples were tested for anti-HBs, anti-HBe and HBV DNA by individual donation NAT. The mean prevalence of anti-HBc was 1.75% in donors that had not been tested for anti-HBc in the past. The percentage of anti-HBs in anti-HBc repeat reactive donors was 93.7%. Samples that were additionally reactive for anti-HBe were anti-HBc reactive in all tested assays. The sample to cut-off (S/Co) values for anti-HBc were lower (competitive assays) in samples that were also positive for anti-HBe, when compared to samples that were only anti-HBc reactive. Most commercially available anti-HBc assays provide sufficient sensitivity for routine screening purposes, and lacking specificity is no longer a serious issue for most of them. Assay differences were recognized for samples that were anti-HBc only reactive. The overall loss of 1.75% of positive testing donors can be significantly reduced to 0.45% by implementation of re-entry procedures for donors with an anti-HBs titre of over 100 IU/l and negative by sensitive ID-NAT.

Effect of the psoralen-based photochemical pathogen inactivation on mitochondrial DNA in platelets.

Photochemical treatment (PCT) of platelet concentrates, using amotosalen HCl and UVA-light, inactivates pathogens by forming adducts between amotosalen and nucleic acids. The impact of the photochemical treatment on pathogens and leukocytes has been studied extensively. Yet little is known about the effect of PCT on nucleic acids in platelets. Platelets contain viable mitochondria and mitochondrial DNA (mtDNA) and this study aimed at evaluating the amotosalen modifications on platelet mtDNA. We applied two independent but complementary molecular assays to investigate qualitative as well as quantitative aspects of the psoralen-mediated DNA modifications in platelet mtDNA. The amotosalen-DNA modification density was measured using (14)C-labeled amotosalen. Amotosalen (150 microM) yielded 4.0 +/- 1.2 psoralen adducts per 1,000 bp in mtDNA after irradiation with 3 J/cm(2) UVA. Furthermore, we tested if the PCT-induced DNA modifications could be detected by a PCR assay. On the basis of PCR inhibition due to amotosalen-DNA adducts, mtDNA-specific PCR assays were developed and tested for their specificity and sensitivity. Our data revealed that mtDNA in platelets is substantially modified by PCT and that these modifications can be documented by a PCR inhibition system.

Implementation of the INTERCEPT Blood System for Platelets into routine blood bank manufacturing procedures: evaluation of apheresis platelets.

BACKGROUND AND OBJECTIVES: The INTERCEPT Blood System for Platelets utilizes amotosalen-HCl (S-59) in combination with ultraviolet A (UVA) light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet concentrates (PCs). To facilitate implementation of this technique into routine blood bank manufacturing procedures, this study evaluated the impact of different time settings of photochemical treatment on in vitro platelet function. MATERIALS AND METHODS: Platelets derived from apheresis (6.5-7.0 x 10(11) platelets) were resuspended in 240 ml of autologous plasma and 360 ml of platelet additive solution (PAS III) and split into two equal-sized PC units. Whereas one unit was not treated, the other was treated with 150 microm amotosalen and 3 J/cm2 UVA light followed by a compound adsorption device (CAD) step for reduction of residual amotosalen and photoproducts. In a first series of experiments (arm A, n = 7), PC units were photochemically treated after an overnight storage period of 16-23 h followed by a CAD step of 4 h. In a second series (arm B, n = 8), photochemical treatment occurred after a short storage time of 4 h with a subsequent CAD step of 16 h. Platelet function was evaluated by assaying blood gas analysis, glucose and lactate concentration, lactate dehydrogenase (LDH), hypotonic shock response (HSR) and the expression of CD62p, over a period of 7 days. RESULTS: Neither of the photochemical treatment procedures showed differences for pH, pCO2, pO2, HCO3, glucose consumption or platelet activation until the end of day 7. Increased lactate values detected for the treated units of arm A at the end of the storage period were independent from the PCT time setting. CONCLUSIONS: Photochemical pathogen inactivation with different initial resting periods between 4 and 23 h, and different CAD steps of 4 and 16 h, had no influence on the platelet in vitro function during 7 days of storage.

The effect of moderate hypovolaemia on microcirculation in healthy older blood donors.

Compensation for acute blood loss requires cardiovascular integrity. In older people, asymptomatic cardiovascular impairment is common. In these subjects, even moderate blood loss is often treated by external volume replacement although its benefits are not clear. We investigated the effect of 450 ml of blood loss on the microcirculation and compensatory mechanisms in healthy older blood donors. Red blood cell count, plasma viscosity and protein concentration were measured. Plasma volume replacement was calculated using haematocrit. We measured microcirculation tissue perfusion by laser Doppler fluxmetry prior to, during and after blood donation. Blood loss was immediately accompanied by a median rapid water shift of 208 ml (interquartile range 134-298 ml). Haemodilution led to a decrease in haematocrit, protein and plasma viscosity. We observed no changes in cutaneous microcirculation. Moderate blood loss is tolerated in older cardiovascularly asymptomatic patients without having an impact on microcirculation. This may reduce the need for external volume replacement.

The flow rate significantly influences the leukocyte depletion rate during prestorage in-line filtration of platelet concentrates.

BACKGROUND: White cell reduction of blood products minimizes the risks of alloimmunization against HLA-antigens, the transmission of viral diseases and the incidence of platelet transfusion reactions. One modern strategy is leukocyte depletion with an integrated filter system immediately after preparation and prior to storage. STUDY DESIGN AND METHODS: We evaluated the efficiency of a novel in-line filter system Sepacell PLX-5 BPS for leukocyte reduction of platelet concentrates (PC) from pooled buffy-coats. A total of 44 PCs were investigated with regard to different filtration flow rates (25-110 ml/min) and leukocyte depletion and thrombocyte recovery rates were analysed. Furthermore, we studied the influence of filtration on PCs over a storage period of 6 days (n = 12) by investigation of pH, lactate and glucose. Platelet function was determined by means of hypotonic shock response, external shape change and expression of CD62p. RESULTS: The mean leukocyte depletion rate was > log 5. After filtration the mean leukocyte count was 0.12 +/- 0.21 x 10(6). In 60% of the PCs the leukocyte count lay below the detection level of the Nageotte chamber, which is < 0.3 x 10(5). The flow rate correlates significantly with the leukocyte count in the PCs (r = 0.325; p = 0.033) and therefore with the leukocyte depletion rate (r = -0.422; p = 0.01). Flow rates under 40 ml lead to a significantly lower leukocyte contamination. Only in one PC, at a flow rate of 84 ml/min, was the leukocyte threshold of 1 x 10(6) exceeded. We did not find a significant correlation between filtration flow rate and thrombocyte recovery (r = 0.315; p = 0.069). The mean platelet count in the PC was 2.88 +/- 0.47 x 10(11). Compared with the thrombocyte count in the pooled buffy coat, the recovery was 68.6%. We observed a decrease of pH, glucose, external shape change and hypotonic shock response over the storage period while lactate and the expression of CD62p increased. CONCLUSION: The filter system Sepacell PLX-5 BPS proved to be suitable for in-line filtration of platelet concentrates prior to storage. Filtration flow rates of up to 40 ml/min allowed efficient leukocyte depletion without significant loss in the quality of the platelet concentrates and the platelet function in vitro.

Effects of blood donation on the physical fitness and hemorheology of healthy elderly donors.

BACKGROUND: International regulations for blood donation recommend a maximum donor age of 65 years. As the average population age is steadily rising in western societies, a considerable group of volunteers is lost to the donor base. STUDY DESIGN AND METHODS: In a prospective study we investigated the effect of a 450-ml whole blood donation on the physical fitness and hemorheology of regular elderly allogeneic blood donors (n = 24, aged 63-69 years, mean = 65). Results were compared with a younger group of regular donors (n = 23, aged 55-62 years, mean = 58) and a group of elderly subjects (n = 7, aged 63-66 years, mean = 65), who did not donate blood for this study. Assessing the physical fitness, we determined the submaximal physical working capacity at a heart rate of 130 min-1 (PWC 130) and the maximal working capacity (MWC) by treadmill exercise testing the day before (day -1) and after donation (d +1). The impact of the blood loss on hemorheology was examined by analyzing the plasma viscosity before, during and after donation. RESULTS: We found an increase of mean values of PWC 130 and MWC on day +1 in all study groups, but increases were only significant in the younger group (PWC 130 p = 0.03; MWC p = 0.04). Values did not differ significantly between the three groups. Plasma viscosity decreased significantly directly after donation in both groups of donors. CONCLUSION: A single blood donation did not alter the physical fitness of otherwise healthy elderly people. The older blood donors and the younger controls showed a similar compensation mechanism to blood loss. We found no general reason for disqualifying blood donors aged 65 years from donating.

[Effect of whole blood preparation and leukocyte filtration on storage of erythrocyte concentrates over 42 days]. / Einfluss von Vollblutpräparation und Leukozytenfiltration auf die Lagerung von Erythrozytenkonzentraten über 42 Tage.

Leukocyte reduction prior to storage of red cell concentrates (RCC) may reduce the incidence of HLA alloimmunization and may improve the quality of stored RCC. We tested an RCC leukoreduction filter system (Baxter) with an integrated Pall RCM-1 filter and investigated the filtration efficiency and the impact on red cells during storage for 42 days after different whole-blood preparation procedures. After whole-blood donation, all units (n = 9, +6 unfiltered controls per group) were either stored at 22 degrees C for up to 6 h (groups 1, 2, 3) or for 24 h (group 4) RCC were either prepared from a triple blood bag system (PL 146, groups 1, 2) or were buffy-coat-depleted (PL 2209, groups 3, 4). Groups 1, 3 und 4 were filtered immediately, whereas group 2 was stored another 18 h at 4 degrees C before filtration. Filtration efficiency and filtration time were determined. Hemolysis, ATP, 2,3-diphosphoglycerate (2,3-DPG), glucose, electrolytes, lactate, hematocrit, Hb and pH were quantified weekly. White blood cells (WBC) were reduced by 3-4 log10 to 0.1-0.3 x 10(6) (mean) by filtration (all groups) regardless of the RCC preparation. Mean filtration times were 1 h 36 min, 33 min, 29 min, and 18 min for the groups 1 to 4, respectively. There were no major differences for the in vitro storage values except for hemolysis and pH, which were elevated in all filtered units, and potassium, which was elevated in the unifiltered units. In conclusion, prestorage leukocyte filtration of RCC reduced the WBC by 3-4 log10, whereas the extended filtration time was a major disadvantage.

Impact of various red cell concentrate preparation methods on the efficiency of prestorage white cell filtration and on red cells during storage for 42 days.

BACKGROUND: White cell (WBC) reduction prior to storage of red cell (RBC) concentrates may reduce the incidence of HLA alloimmunization and may improve the quality of stored RBCs. STUDY DESIGN AND METHODS: An integrated WBC-reduction filter system was tested after various RBC preparation procedures (from whole blood), and the influence of filtration on RBCs during storage for 42 days was investigated. Four additive system RBC preparation protocols were used. Units prepared from conventional triple blood bags were held for 4 to 6 hours at 22 degrees C, and then the RBCs were separated via a hard spin and filtration, performed immediately (Group 1) or after 18 hours' storage at 4 degrees C (Group 2). Units prepared from a top-and-bottom collection system were held at 22 degrees C for 4 to 6 or 22 to 24 hours; the centrifuged RBCs were filtered immediately after preparation (Groups 3 and 4, respectively, by holding time). WBC reduction and filtration time were analyzed. The impact of WBC filtration on pH, hemolysis rate, hemoglobin content, ATP, potassium glucose, and lactate was investigated weekly during storage for 42 days. RESULTS: Filtration reduced the mean WBC count by 3 to 4 log10, to 0.19 +/- 0.25 x 10(6), regardless of the RBC preparation method. Mean filtration times differed significantly between the groups and were longest for Group 1. Besides hemolysis and pH values, which were greater in all filtered units, no major differences were found in filtered and unfiltered RBCs during the storage interval. CONCLUSION: The efficiency of prestorage WBC filtration of RBCs was unaffected by the preparation procedure. However, the filtration time for RBCs freshly prepared in the conventional triple blood bag system without buffy-coat depletion was unacceptable. No major metabolic differences between filtered and unfiltered RBCs during 42 days of storage were found.

[Blood donation after reaching 65 years of age]. / Blutspende nach Erreichen des 65. Lebensjahres.

European regulations for blood donation recommend a maximum donor age of 65 years. On the other hand, the percentage of the population in this age group is rapidly increasing in Western countries, and in autologous blood donation programs this limitation has already been abandoned. In a prospective study we examined blood donation in elderly donors (18 male and 5 female; mean age 65 years, range 64-69) in comparison to a younger control group (15/9; 58, 55-63). All donors were regular blood donors and had donated for at least 3 years. We investigated the exercise capacity before and after donation of 450 ml whole blood by examination of the physical working capacity (PWC) at heart rates of 110/min and 130/min through treadmill exercise testing and determined the blood viscosity. Additionally, whole-blood count, hemoglobin, plasma-ferritin levels and total iron binding capacity were measured immediately after donation and on days 7, 28 and 49. We found a decrease in whole-blood viscosity and a moderate increase in PWC at heart rates of 110/min and 130/min after donation in both groups. Red cell count and values of hemoglobin and ferritin were significantly lower in both groups after donation and returned to pre-donation values by day 49 in the younger control group. We detected no deterioration in exercise capacity after whole-blood donation in elderly blood donors over 65 years when compared with a younger control group. We suggest that blood donation in otherwise healthy persons aged over 65 years should be accepted.